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SUMMARY:The limited lifespan of human microvascular endothelial cells in cell culture represents a major obstacle for the study of microvascular pathobiology. To date, no endothelial cell line is available that demonstrates all of the fundamental characteristics of microvascular endothelial cells. We have generated endothelial cell lines from human pulmonary microvascular endothelial cells (HPMEC) isolated from adult donors. HPMEC were cotransfected with a plasmid encoding the catalytic component of telomerase (hTERT) and a plasmid encoding the simian virus 40 (SV40) large T antigen. Cells transfected with either plasmid alone had an extended lifespan, but the cultures eventually entered crisis after several months of proliferation. Only those cells that were transfected with both plasmids acquired the capacity to grow in vitro without demonstrating major crisis, and these cells have been in culture for 24 months. HPMEC isolated from two different donors were used, generating two populations of immortalized cells, HPMEC-ST1 and HPMEC-ST2. Single cell-derived clones of the immortalized cells HPMEC-ST1 exhibited growth characteristics that were similar to those of the parental HPMEC. One selected clone, HPMEC-ST1.6R, displayed all major constitutively expressed and inducible endothelial phenotypic markers, including platelet endothelial cell adhesion molecule (PECAM-1, CD31), von Willebrand factor (vWF), and the adhesion molecules, intercellular adhesion molecule (ICAM-1), vascular adhesion molecule (VCAM-1), and E-selectin. In addition, an angiogenic response was demonstrated by sprout formation on a biological extracellular matrix (Matrigel). The HPMEC-ST1.6R cells did not form tumors in nude mice. The microvascular endothelial cell line, HPMEC-ST1.6R, will be a valuable tool for the study of microvascular endothelial physiology and pathology including gene expression, angiogenesis, and tumorigenesis. (Lab Invest 2001, 81:1717-1727.

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Objectives-Sphingosine 1-phosphate (S1P) is a bioactive phospholipid acting both as a ligand for the G protein-coupled receptors S1P 1-5 and as a second messenger. Because S1P 1 knockout is lethal in the transgenic mouse, an alternative approach to study the function of S1P 1 in endothelial cells is needed. Methods and Results-All human endothelial cells analyzed expressed abundant S1P 1 transcripts. We permanently silenced (by RNA interference) the expression of S1P 1 in the human endothelial cell lines AS-M.5 and ISO-HAS.1. The S1P 1 knock-down cells manifested a distinct morphology and showed neither actin ruffles in response to S1P nor an angiogenic reaction. In addition, these cells were more sensitive to oxidant stress-mediated injury. New S1P 1 -dependent gene targets were identified in human endothelial cells. S1P 1 silencing decreased the expression of platelet-endothelial cell adhesion molecule-1 and VE-cadherin and abolished the induction of E-selectin after cell stimulation with lipopolysaccharide or tumor necrosis factor-␣. Microarray analysis revealed downregulation of further endothelial specific transcripts after S1P 1 silencing. Conclusions-Long-term silencing of S1P 1 enabled us for the first time to demonstrate the involvement of S1P 1 in key functions of endothelial cells and to identify new S1P 1 -dependent gene targets. Key Words: S1P 1 Ⅲ functional analysis Ⅲ siRNA Ⅲ permanent inhibition Ⅲ endothelial cells T he bioactive sphingolipid metabolite, sphingosine 1-phosphate (S1P), is an important component of serum that is released primarily from activated platelets. S1P is a multifunctional physiologic mediator implicated in the regulation of a broad spectrum of biologic processes, including proliferation, survival, regulation of cytoskeletal reorganization, motility, and differentiation in many cell types. [1][2][3][4][5] The response of cells to S1P has been shown to be mediated predominantly by G protein-coupled receptors. Five receptors encoded by the endothelial differentiation gene family that bind S1P with high affinity have been described. 6 -10 The receptors couple to multiple G proteins that activate different intracellular signaling pathways. Several of these receptors are simultaneously coexpressed on the same cell.In addition to acting as a ligand of cell surface receptors, S1P can function as a second messenger. 3,4,11 A variety of external stimuli, particularly growth factors and chemoattractants, as well as lysophosphatidic acid and S1P, have been reported to strongly stimulate S1P kinase (SphK) to generate intracellular S1P, which can mobilize calcium from internal stores, 12 regulate cell survival by activating the transcription factor nuclear factor-B, 13 and control cell proliferation by mediating Ras and extracellular signal-regulated kinase 1/2 activation in cells stimulated with vascular endothelial growth factor (VEGF). 14 Stimulation of the S1P 1 receptor (previously known as endothelial differentiation gene-1, edg-1) activates a G ilinked pathway leading to cell gro...